The Biochemistry of Ferritin 205 



and when administered intravenously to the dog for study of its anti- 

 diuretic effect. 



(b) Ferritin can be separated into a number of fractions which have 

 varying total iron: total nitrogen ratios, all of which, however, are 

 equally active on the basis of nitrogen content. 



The first of these results could be explained without eliminating 

 iron as a participant if it could be shown that apoferritin, on intra- 

 venous injection, combines with iron in the plasma to yield active 

 ferritin. The second could be explained if the various fractions ob- 

 tained by fractionating ferritin could be shown to consist of two forms 

 of iron, one of which was present in these fractions in a constant 

 ratio to the nitrogen content. This second form of iron would fulfill 

 the requirements of oxidation-reduction reactions if it were ionic and 

 therefore capable of existence either in the ferric or ferrous state. 



Iodoacetamide-treated ferritin, with no measurable sulfhydryl con- 

 tent was incubated with cysteine, ascorbic acid, or reduced glutathione. 

 The reagents were then removed by dialysis and the ferritin analyzed 

 for sulfhydryl groups. No sulfhydryl groups were regenerated by 

 cysteine or ascorbic acid, but a definite though small increase appeared 

 after treatment with glutathione. The new sulfhydryl groups were, 

 however, part of the added glutathione which had been bound to the 

 ferritin molecule. This was established by incubating iodoacetamide 

 ferritin with S 35 -labeled glutathione followed by extensive dialysis 

 against water. The radioactivity associated with the ferritin was 

 found to be approximately equal to the sulfhydryl content as measured 

 chemically by reaction with iodoacetamide and could be removed by 

 treatment with trichloracetic acid. These results make it unlikely 

 that ferritin activity is directly concerned with sulfhydryl groups. 



Active ferritin solutions were now treated with a,a'-dipyridyl and 

 the ferritin precipitated by addition of an equal volume of saturated 

 ammonium sulfate. The clear protein-free supernatant solution had 

 the typical pink color of the ferrous-dipyridyl complex even when the 

 reaction was carried out at pH 7.4. At a constant concentration of 

 dipyridyl and varying concentrations of ferritin the data showed that, 

 as the ferritin was diluted, a larger fraction of its total iron was bound 

 by dipyridyl in the form of ferrous iron. At a constant concentration 

 of both dipyridyl and ferritin the quantity of ferrous iron bound by 

 dipyridyl increased with decrease in pH. These results were consistent 

 with the hypothesis of a competition between dipyridyl and ferritin for 

 its ferrous iron, which was therefore capable of some dissociation and 

 probably at or near the surface of the protein. 



